Pseudomonas aeruginosa surface motility and invasion into competing communities enhances interspecies antagonism.

Chronic polymicrobial infections involving Pseudomonas aeruginosa and Staphylococcus aureus are prevalent, difficult to eradicate, and associated with poor health outcomes. Therefore, understanding interactions between these pathogens is important to inform improved treatment development. We previously demonstrated that P. aeruginosa is attracted to S. aureus using type IV pili-mediated chemotaxis, but the impact of attraction on S. aureus growth and physiology remained unknown. Using live single-cell confocal imaging to visualize microcolony structure, spatial organization, and survival of S. aureus during coculture, we found that interspecies chemotaxis provides P. aeruginosa a competitive advantage by promoting invasion into and disruption of S. aureus microcolonies. This behavior renders S. aureus susceptible to P. aeruginosa antimicrobials. Conversely, in the absence of type IV pilus motility, P. aeruginosa cells exhibit reduced invasion of S. aureus colonies. Instead, P. aeruginosa builds a cellular barrier adjacent to S. aureus and secretes diffusible, bacteriostatic antimicrobials like 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) into the S. aureus colonies. P. aeruginosa reduced invasion leads to the formation of denser and thicker S. aureus colonies with significantly increased HQNO-mediated lactic acid fermentation, a physiological change that could complicate the effective treatment of infections. Finally, we show that P. aeruginosa motility modifications of spatial structure enhance competition against S. aureus. Overall, these studies build on our understanding of how P. aeruginosa type IV pili-mediated interspecies chemotaxis mediates polymicrobial interactions, highlighting the importance of spatial positioning in mixed-species communities.

In utero and postnatal ivacaftor/lumacaftor therapy rescues multiorgan disease in CFTR-F508del ferrets.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene, with F508del being the most prevalent mutation. The combination of CFTR modulators (potentiator and correctors) has provided benefit to CF patients carrying the F508del mutation; however, the safety and effectiveness of in utero combination modulator therapy remains unclear. We created a F508del ferret model to test whether ivacaftor/lumacaftor (VX-770/VX-809) therapy can rescue in utero and postnatal pathologies associated with CF. Using primary intestinal organoids and air-liquid interface cultures of airway epithelia, we demonstrate that the F508del mutation in ferret CFTR results in a severe folding and trafficking defect, which can be partially restored by treatment with CFTR modulators. In utero treatment of pregnant jills with ivacaftor/lumacaftor prevented meconium ileus at birth in F508del kits and sustained postnatal treatment of CF offspring improved survival and partially protected from pancreatic insufficiency. Withdrawal of ivacaftor/lumacaftor treatment from juvenile CF ferrets reestablished pancreatic and lung diseases, with altered pulmonary mechanics. These findings suggest that in utero intervention with a combination of CFTR modulators may provide therapeutic benefits to individuals with F508del. This CFTR-F508del ferret model may be useful for testing therapies using clinically translatable endpoints.

The type IV pilus chemoreceptor PilJ controls chemotaxis of one bacterial species towards another.

Bacteria live in social communities, where the ability to sense and respond to interspecies and environmental signals is critical for survival. We previously showed the pathogen Pseudomonas aeruginosa detects secreted peptides from bacterial competitors and navigates through interspecies signal gradients using pilus-based motility. Yet, it was unknown whether P. aeruginosa utilizes a designated chemosensory system for this behavior. Here, we performed a systematic genetic analysis of a putative pilus chemosensory system, followed by high-speed live-imaging and single-cell tracking, to reveal behaviors of mutants that retain motility but are blind to interspecies signals. The enzymes predicted to methylate (PilK) and demethylate (ChpB) the putative pilus chemoreceptor, PilJ, are necessary for cells to control the direction of migration. While these findings implicate PilJ as a bona fide chemoreceptor, such function had yet to be experimentally defined, as full-length PilJ is essential for motility. Thus, we constructed systematic genetic modifications of PilJ and found that without the predicted ligand binding domains or predicted methylation sites, cells lose the ability to detect competitor gradients, despite retaining pilus-mediated motility. Chemotaxis trajectory analysis revealed that increased probability and size of P. aeruginosa pilus-mediated steps towards S. aureus peptides, versus steps away, determines motility bias in wild type cells. However, PilJ mutants blind to interspecies signals take less frequent steps towards S. aureus or steps of equal size towards and away. Collectively, this work uncovers the chemosensory nature of PilJ, provides insight into how cell movements are biased during pilus-based chemotaxis, and identifies chemotactic interactions necessary for bacterial survival in polymicrobial communities, revealing putative pathways where therapeutic intervention might disrupt bacterial communication.

The great divide: rhamnolipids mediate separation between P. aeruginosa and S. aureus.

The interactions between bacterial species during infection can have significant impacts on pathogenesis. Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic bacterial pathogens that can co-infect hosts and cause serious illness. The factors that dictate whether one species outcompetes the other or whether the two species coexist are not fully understood. We investigated the role of surfactants in the interactions between these two species on a surface that enables P. aeruginosa to swarm. We found that P. aeruginosa swarms are repelled by colonies of clinical S. aureus isolates, creating physical separation between the two strains. This effect was abolished in mutants of S. aureus that were defective in the production of phenol-soluble modulins (PSMs), which form amyloid fibrils around wild-type S. aureus colonies. We investigated the mechanism that establishes physical separation between the two species using Imaging of Reflected Illuminated Structures (IRIS), which is a non-invasive imaging method that tracks the flow of surfactants produced by P. aeruginosa. We found that PSMs produced by S. aureus deflected the surfactant flow, which in turn, altered the direction of P. aeruginosa swarms. These findings show that rhamnolipids mediate physical separation between P. aeruginosa and S. aureus, which could facilitate coexistence between these species. Additionally, we found that a number of molecules repelled P. aeruginosa swarms, consistent with a surfactant deflection mechanism. These include Bacillus subtilis surfactant, the fatty acids oleic acid and linoleic acid, and the synthetic lubricant polydimethylsiloxane. Lung surfactant repelled P. aeruginosa swarms and inhibited swarm expansion altogether at higher concentration. Our results suggest that surfactant interactions could have major impacts on bacteria-bacteria and bacteria-host relationships. In addition, our findings uncover a mechanism responsible for P. aeruginosa swarm development that does not rely solely on sensing but instead is based on the flow of surfactant.

Staphylococcal secreted cytotoxins are competition sensing signals for Pseudomonas aeruginosa.

Coinfection with two notorious opportunistic pathogens, the Gram-negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus , dominates chronic pulmonary infections. While coinfection is associated with poor patient outcomes, the interspecies interactions responsible for such decline remain unknown. Here, we dissected molecular mechanisms of interspecies sensing between P. aeruginosa and S. aureus . We discovered that P. aeruginosa senses S. aureus secreted peptides and, counterintuitively, moves towards these toxins. P. aeruginosa tolerates such a strategy through "competition sensing", whereby it preempts imminent danger/competition by arming cells with type six secretion (T6S) and iron acquisition systems. Intriguingly, while T6S is predominantly described as weaponry targeting Gram-negative and eukaryotic cells, we find that T6S is essential for full P. aeruginosa competition with S. aureus , a previously undescribed role for T6S. Importantly, competition sensing was activated during coinfection of bronchial epithelia, including T6S islands targeting human cells. This study reveals critical insight into both interspecies competition and how antagonism may cause collateral damage to the host environment.

Sustained Coinfections with Staphylococcus aureus and Pseudomonas aeruginosa in Cystic Fibrosis.

Rationale: Staphylococcus aureus and Pseudomonas aeruginosa often infect the airways in cystic fibrosis (CF). Because registry studies show higher prevalence of P. aeruginosa versus S. aureus in older patients with CF, a common assumption is that P. aeruginosa replaces S. aureus over time. In vitro, P. aeruginosa can outgrow and kill S. aureus. However, it is unknown how rapidly P. aeruginosa replaces S. aureus in patients with CF.Methods: We studied a longitudinal cohort of children and adults with CF who had quantitative sputum cultures. We determined the abundance of P. aeruginosa and S. aureus in cfu/ml. We determined the duration and persistence of infections and measured longitudinal changes in culture positivity and abundance for each organism.Measurements and Main Results: Between 2004 and 2017, 134 patients had ≥10 quantitative cultures, with median observation time of 10.15 years. One hundred twenty-four patients had at least one positive culture for P. aeruginosa, and 123 had at least one positive culture for S. aureus. Both species had median abundance of >106 cfu/ml. Culture abundance was stable over time for both organisms. There was an increase in the prevalence of S. aureus/P. aeruginosa coinfection but no decrease in S. aureus prevalence within individuals over time.Conclusions: S. aureus and P. aeruginosa are abundant in CF sputum cultures. Contrary to common assumption, we found no pattern of replacement of S. aureus by P. aeruginosa. Many patients with CF have durable long-term coinfection with these organisms. New strategies are needed to prevent and treat these infections.

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time. Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

Exogenous Alginate Protects Staphylococcus aureus from Killing by Pseudomonas aeruginosa.

Cystic fibrosis (CF) patients chronically infected with both Pseudomonas aeruginosa and Staphylococcus aureus have worse health outcomes than patients who are monoinfected with either P. aeruginosa or S. aureus We showed previously that mucoid strains of P. aeruginosa can coexist with S. aureusin vitro due to the transcriptional downregulation of several toxic exoproducts typically produced by P. aeruginosa, including siderophores, rhamnolipids, and HQNO (2-heptyl-4-hydroxyquinoline N-oxide). Here, we demonstrate that exogenous alginate protects S. aureus from P. aeruginosa in both planktonic and biofilm coculture models under a variety of nutritional conditions. S. aureus protection in the presence of exogenous alginate is due to the transcriptional downregulation of pvdA, a gene required for the production of the iron-scavenging siderophore pyoverdine as well as the downregulation of the PQS (Pseudomonas quinolone signal) (2-heptyl-3,4-dihydroxyquinoline) quorum sensing system. The impact of exogenous alginate is independent of endogenous alginate production. We further demonstrate that coculture of mucoid P. aeruginosa with nonmucoid P. aeruginosa strains can mitigate the killing of S. aureus by the nonmucoid strain of P. aeruginosa, indicating that the mechanism that we describe here may function in vivo in the context of mixed infections. Finally, we investigated a panel of mucoid clinical isolates that retain the ability to kill S. aureus at late time points and show that each strain has a unique expression profile, indicating that mucoid isolates can overcome the S. aureus-protective effects of mucoidy in a strain-specific manner.IMPORTANCE CF patients are chronically infected by polymicrobial communities. The two dominant bacterial pathogens that infect the lungs of CF patients are P. aeruginosa and S. aureus, with ∼30% of patients coinfected by both species. Such coinfected individuals have worse outcomes than monoinfected patients, and both species persist within the same physical space. A variety of host and environmental factors have been demonstrated to promote P. aeruginosa-S. aureus coexistence, despite evidence that P. aeruginosa kills S. aureus when these organisms are cocultured in vitro Thus, a better understanding of P. aeruginosa-S. aureus interactions, particularly mechanisms by which these microorganisms are able to coexist in proximal physical space, will lead to better-informed treatments for chronic polymicrobial infections.

High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates.

BACKGROUND: Staphylococcus aureus is a highly prevalent respiratory pathogen in cystic fibrosis (CF). It is unclear how this organism establishes chronic infections in CF airways. We hypothesized that S. aureus isolates from patients with CF would share common virulence properties that enable chronic infection. METHODS: 77 S. aureus isolates were obtained from 45 de-identified patients with CF at the University of Iowa. We assessed isolates phenotypically and used genotyping assays to determine the presence or absence of 18 superantigens (SAgs). RESULTS: We observed phenotypic diversity among S. aureus isolates from patients with CF. Genotypic analysis for SAgs revealed 79.8% of CF clinical isolates carried all six members of the enterotoxin gene cluster (EGC). MRSA and MSSA isolates had similar prevalence of SAgs. We additionally observed that EGC SAgs were prevalent in S. aureus isolated from two geographically distinct CF centers. CONCLUSIONS: S. aureus SAgs belonging to the EGC are highly prevalent in CF clinical isolates. The greater prevalence in these SAgs in CF airway specimens compared to skin isolates suggests that these toxins confer selective advantage in the CF airway.

Interspecies interactions induce exploratory motility in Pseudomonas aeruginosa.

Microbes often live in multispecies communities where interactions among community members impact both the individual constituents and the surrounding environment. Here, we developed a system to visualize interspecies behaviors at initial encounters. By imaging two prevalent pathogens known to be coisolated from chronic illnesses, Pseudomonas aeruginosa and Staphylococcus aureus, we observed P. aeruginosa can modify surface motility in response to secreted factors from S. aureus. Upon sensing S. aureus, P. aeruginosa transitioned from collective to single-cell motility with an associated increase in speed and directedness - a behavior we refer to as 'exploratory motility'. Explorer cells moved preferentially towards S. aureus and invaded S. aureus colonies through the action of the type IV pili. These studies reveal previously undescribed motility behaviors and lend insight into how P. aeruginosa senses and responds to other species. Identifying strategies to harness these interactions may open avenues for new antimicrobial strategies.

Frontline Science: Pathological conditioning of human neutrophils recruited to the airway milieu in cystic fibrosis.

Recruitment of neutrophils to the airways, and their pathological conditioning therein, drive tissue damage and coincide with the loss of lung function in patients with cystic fibrosis (CF). So far, these key processes have not been adequately recapitulated in models, hampering drug development. Here, we hypothesized that the migration of naïve blood neutrophils into CF airway fluid in vitro would induce similar functional adaptation to that observed in vivo, and provide a model to identify new therapies. We used multiple platforms (flow cytometry, bacteria-killing, and metabolic assays) to characterize functional properties of blood neutrophils recruited in a transepithelial migration model using airway milieu from CF subjects as an apical chemoattractant. Similarly to neutrophils recruited to CF airways in vivo, neutrophils migrated into CF airway milieu in vitro display depressed phagocytic receptor expression and bacterial killing, but enhanced granule release, immunoregulatory function (arginase-1 activation), and metabolic activities, including high Glut1 expression, glycolysis, and oxidant production. We also identify enhanced pinocytic activity as a novel feature of these cells. In vitro treatment with the leukotriene pathway inhibitor acebilustat reduces the number of transmigrating neutrophils, while the metabolic modulator metformin decreases metabolism and oxidant production, but fails to restore bacterial killing. Interestingly, we describe similar pathological conditioning of neutrophils in other inflammatory airway diseases. We successfully tested the hypothesis that recruitment of neutrophils into airway milieu from patients with CF in vitro induces similar pathological conditioning to that observed in vivo, opening new avenues for targeted therapeutic intervention.

Mixed Communities of Mucoid and Nonmucoid Pseudomonas aeruginosa Exhibit Enhanced Resistance to Host Antimicrobials.

Pseudomonas aeruginosa causes chronic pulmonary infections in patients with cystic fibrosis (CF). P. aeruginosa mucoid conversion, defined by overproduction of the exopolysaccharide alginate, correlates with accelerated decline in CF patient lung function. Recalcitrance of the mucoid phenotype to clearance by antibiotics and the immune response is well documented. However, despite advantages conferred by mucoidy, mucoid variants often revert to a nonmucoid phenotype both in vitro and in vivo Mixed populations of mucoid isolates and nonmucoid revertants are recovered from CF lungs, suggesting a selective benefit for coexistence of these variants. In this study, cocultures of mucoid and nonmucoid variants exhibited enhanced resistance to two host antimicrobials: LL-37, a cationic antimicrobial peptide, and hydrogen peroxide (H2O2). Alginate production by mucoid isolates protected nonmucoid variants in consortia from LL-37, as addition of alginate exogenously to nonmucoid variants abrogated LL-37 killing. Conversely, nonmucoid revertants shielded mucoid variants from H2O2 stress via catalase (KatA) production, which was transcriptionally repressed by AlgT and AlgR, central regulators of alginate biosynthesis. Furthermore, extracellular release of KatA by nonmucoid revertants was dependent on lys, encoding an endolysin implicated in autolysis and extracellular DNA (eDNA) release. Overall, these data provide a rationale to study interactions of P. aeruginosa mucoid and nonmucoid variants as contributors to evasion of innate immunity and persistence within the CF lung.IMPORTANCEP. aeruginosa mucoid conversion within lungs of cystic fibrosis (CF) patients is a hallmark of chronic infection and predictive of poor prognosis. The selective benefit of mixed populations of mucoid and nonmucoid variants, often isolated from chronically infected CF patients, has not been explored. Here, we show that mixed-variant communities of P. aeruginosa demonstrate advantages in evasion of innate antimicrobials via production of shared goods: alginate and catalase. These data argue for therapeutically targeting multiple constituents (both mucoid and nonmucoid variants) within diversified P. aeruginosa communities in vivo, as these variants can differentially shield one another from components of the host response.

Pseudomonas aeruginosa Alginate Overproduction Promotes Coexistence with Staphylococcus aureus in a Model of Cystic Fibrosis Respiratory Infection.

While complex intra- and interspecies microbial community dynamics are apparent during chronic infections and likely alter patient health outcomes, our understanding of these interactions is currently limited. For example, Pseudomonas aeruginosa and Staphylococcus aureus are often found to coinfect the lungs of patients with cystic fibrosis (CF), yet these organisms compete under laboratory conditions. Recent observations that coinfection correlates with decreased health outcomes necessitate we develop a greater understanding of these interbacterial interactions. In this study, we tested the hypothesis that P. aeruginosa and/or S. aureus adopts phenotypes that allow coexistence during infection. We compared competitive interactions of P. aeruginosa and S. aureus isolates from mono- or coinfected CF patients employing in vitro coculture models. P. aeruginosa isolates from monoinfected patients were more competitive toward S. aureus than P. aeruginosa isolates from coinfected patients. We also observed that the least competitive P. aeruginosa isolates possessed a mucoid phenotype. Mucoidy occurs upon constitutive activation of the sigma factor AlgT/U, which regulates synthesis of the polysaccharide alginate and dozens of other secreted factors, including some previously described to kill S. aureus Here, we show that production of alginate in mucoid strains is sufficient to inhibit anti-S. aureus activity independent of activation of the AlgT regulon. Alginate reduces production of siderophores, 2-heptyl-4-hydroxyquinolone-N-oxide (HQNO), and rhamnolipids-each required for efficient killing of S. aureus These studies demonstrate alginate overproduction may be an important factor driving P. aeruginosa coinfection with S. aureusIMPORTANCE Numerous deep-sequencing studies have revealed the microbial communities present during respiratory infections in cystic fibrosis (CF) patients are diverse, complex, and dynamic. We now face the challenge of determining the influence of these community dynamics on patient health outcomes and identifying candidate targets to modulate these interactions. We make progress toward this goal by determining that the polysaccharide alginate produced by mucoid strains of P. aeruginosa is sufficient to inhibit multiple secreted antimicrobial agents produced by this organism. Importantly, these secreted factors are required to outcompete S. aureus, when the microbes are grown in coculture; thus we propose a mechanism whereby mucoid P. aeruginosa can coexist with S. aureus Finally, the approach used here can serve as a platform to investigate the interactions among other CF pathogens.

Bacterial Extracellular Polysaccharides in Biofilm Formation and Function.

Microbes produce a biofilm matrix consisting of proteins, extracellular DNA, and polysaccharides that is integral in the formation of bacterial communities. Historical studies of polysaccharides revealed that their overproduction often alters the colony morphology and can be diagnostic in identifying certain species. The polysaccharide component of the matrix can provide many diverse benefits to the cells in the biofilm, including adhesion, protection, and structure. Aggregative polysaccharides act as molecular glue, allowing the bacterial cells to adhere to each other as well as surfaces. Adhesion facilitates the colonization of both biotic and abiotic surfaces by allowing the bacteria to resist physical stresses imposed by fluid movement that could separate the cells from a nutrient source. Polysaccharides can also provide protection from a wide range of stresses, such as desiccation, immune effectors, and predators such as phagocytic cells and amoebae. Finally, polysaccharides can provide structure to biofilms, allowing stratification of the bacterial community and establishing gradients of nutrients and waste products. This can be advantageous for the bacteria by establishing a heterogeneous population that is prepared to endure stresses created by the rapidly changing environments that many bacteria encounter. The diverse range of polysaccharide structures, properties, and roles highlight the importance of this matrix constituent to the successful adaptation of bacteria to nearly every niche. Here, we present an overview of the current knowledge regarding the diversity and benefits that polysaccharide production provides to bacterial communities within biofilms.

Cationic antimicrobial peptides promote microbial mutagenesis and pathoadaptation in chronic infections.

Acquisition of adaptive mutations is essential for microbial persistence during chronic infections. This is particularly evident during chronic Pseudomonas aeruginosa lung infections in cystic fibrosis (CF) patients. Thus far, mutagenesis has been attributed to the generation of reactive species by polymorphonucleocytes (PMN) and antibiotic treatment. However, our current studies of mutagenesis leading to P. aeruginosa mucoid conversion have revealed a potential new mutagen. Our findings confirmed the current view that reactive oxygen species can promote mucoidy in vitro, but revealed PMNs are proficient at inducing mucoid conversion in the absence of an oxidative burst. This led to the discovery that cationic antimicrobial peptides can be mutagenic and promote mucoidy. Of specific interest was the human cathelicidin LL-37, canonically known to disrupt bacterial membranes leading to cell death. An alternative role was revealed at sub-inhibitory concentrations, where LL-37 was found to induce mutations within the mucA gene encoding a negative regulator of mucoidy and to promote rifampin resistance in both P. aeruginosa and Escherichia coli. The mechanism of mutagenesis was found to be dependent upon sub-inhibitory concentrations of LL-37 entering the bacterial cytosol and binding to DNA. LL-37/DNA interactions then promote translesion DNA synthesis by the polymerase DinB, whose error-prone replication potentiates the mutations. A model of LL-37 bound to DNA was generated, which reveals amino termini α-helices of dimerized LL-37 bind the major groove of DNA, with numerous DNA contacts made by LL-37 basic residues. This demonstrates a mutagenic role for antimicrobials previously thought to be insusceptible to resistance by mutation, highlighting a need to further investigate their role in evolution and pathoadaptation in chronic infections.

ChIP-Seq and RNA-Seq reveal an AmrZ-mediated mechanism for cyclic di-GMP synthesis and biofilm development by Pseudomonas aeruginosa.

The transcription factor AmrZ regulates genes important for P. aeruginosa virulence, including type IV pili, extracellular polysaccharides, and the flagellum; however, the global effect of AmrZ on gene expression remains unknown, and therefore, AmrZ may directly regulate many additional genes that are crucial for infection. Compared to the wild type strain, a ΔamrZ mutant exhibits a rugose colony phenotype, which is commonly observed in variants that accumulate the intracellular second messenger cyclic diguanylate (c-di-GMP). Cyclic di-GMP is produced by diguanylate cyclases (DGC) and degraded by phosphodiesterases (PDE). We hypothesized that AmrZ limits the intracellular accumulation of c-di-GMP through transcriptional repression of gene(s) encoding a DGC. In support of this, we observed elevated c-di-GMP in the ΔamrZ mutant compared to the wild type strain. Consistent with other strains that accumulate c-di-GMP, when grown as a biofilm, the ΔamrZ mutant formed larger microcolonies than the wild-type strain. This enhanced biofilm formation was abrogated by expression of a PDE. To identify potential target DGCs, a ChIP-Seq was performed and identified regions of the genome that are bound by AmrZ. RNA-Seq experiments revealed the entire AmrZ regulon, and characterized AmrZ as an activator or repressor at each binding site. We identified an AmrZ-repressed DGC-encoding gene (PA4843) from this cohort, which we named AmrZ dependent cyclase A (adcA). PAO1 overexpressing adcA accumulates 29-fold more c-di-GMP than the wild type strain, confirming the cyclase activity of AdcA. In biofilm reactors, a ΔamrZ ΔadcA double mutant formed smaller microcolonies than the single ΔamrZ mutant, indicating adcA is responsible for the hyper biofilm phenotype of the ΔamrZ mutant. This study combined the techniques of ChIP-Seq and RNA-Seq to define the comprehensive regulon of a bifunctional transcriptional regulator. Moreover, we identified a c-di-GMP mediated mechanism for AmrZ regulation of biofilm formation and chronicity.

Mutagenesis by host antimicrobial peptides: insights into microbial evolution during chronic infections.

Antimicrobial peptides (AMPs) are produced by the mammalian immune system to fight invading pathogens. The best understood function of AMPs is to integrate into the membranes of microbes, thereby disrupting and killing cells. However, a recent study [PLoS Pathogens (2014) 10, e1004083] provides evidence that at subinhibitory levels, AMPs promote mutations in bacterial DNA, which enhance bacterial survival. In particular, in the bacterium Pseudomonas aeruginosa, one AMP called LL-37 can promote mutations, which enable the bacteria to overproduce a protective sugar coating, a process called mucoid conversion. P. aeruginosa mucoid conversion is a major risk factor for those suffering from cystic fibrosis (CF), one of the most common lethal, heritable diseases in the US. LL-37 was found to produce mutations by penetrating the bacterial cell and binding to bacterial DNA. It was proposed that LL-37 binding DNA disrupts normal DNA replication and potentiates mutations. Importantly, LL-37 induced mutagenesis was also found to promote resistance to rifampicin in both P. aeruginosa and E. coli. This suggests that AMP-induced mutagenesis may be important for a broad range of chronic diseases and pathogens.

BgaA acts as an adhesin to mediate attachment of some pneumococcal strains to human epithelial cells.

Streptococcus pneumoniae colonization of the respiratory tract is an essential precursor for pneumococcal disease. To colonize efficiently, bacteria must adhere to the epithelial-cell surface. S. pneumoniae possesses surface-associated exoglycosidases that are capable of sequentially deglycosylating human glycans. Two exoglycosidases, neuraminidase (NanA) and ß-galactosidase (BgaA), have previously been shown to contribute to S. pneumoniae adherence to human epithelial cells, as deletion of either of these genes results in reduced adherence. It has been suggested that these enzymes may modulate adherence by cleaving sugars to reveal a receptor on host cells. Pretreatment of epithelial cells with exogenous neuraminidase restores the adherence of a nanA mutant, whereas pretreatment with ß-galactosidase does not restore the adherence of a bgaA mutant. These data suggest that BgaA may not function to reveal a receptor, and implicate an alternative role for BgaA in adherence. Here we demonstrate that ß-galactosidase activity is not required for BgaA-mediated adherence. Addition of recombinant BgaA (rBgaA) to adherence assays and pretreatment of epithelial cells with rBgaA both significantly reduced the level of adherence of the parental strain, but not the BgaA mutant. One possible explanation of these data is that BgaA is acting as an adhesin and that rBgaA is binding to the receptor, preventing bacterial binding. A bead-binding assay demonstrated that BgaA can bind directly to human epithelial cells, supporting the hypothesis that BgaA is an adhesin. Preliminary characterization of the epithelial-cell receptor suggests that it is a glycan in the context of a glycosphingolipid. To further establish the relevance of this adherence mechanism, we demonstrated that BgaA-mediated adherence contributed to adherence of a recent clinical isolate to primary human epithelial cells. Together, these data suggest a novel role for BgaA as an adhesin and suggest that this mechanism could contribute to adherence of at least some pneumococcal strains in vivo.

Identification of a pneumococcal glycosidase that modifies O-linked glycans.

Colonization of the airway by Streptococcus pneumoniae is typically asymptomatic; however, progression of bacteria beyond the oronasopharynx can cause diseases including otitis media and pneumonia. The mechanisms by which S. pneumoniae establishes and maintains colonization remain poorly understood. Both N-linked and O-linked glycans are abundant in the airway. Our previous research demonstrated that S. pneumoniae can sequentially deglycosylate N-linked glycans and suggested that this modification of sugar structures may aid in colonization. There is published evidence that S. pneumoniae expresses a secreted O-glycosidase that cleaves galactose beta1-3 N-acetylgalactosamine (Galbeta1-3GalNAc) from core-1 O-linked glycans; however, the biological function of this enzyme has not previously been determined. We established that the activity is not secreted but is instead surface associated in a sortase-dependent manner. Genome analysis revealed an open reading frame predicted to encode a sortase-dependent surface protein with sequence similarity to the O-glycosidase of Bifidobacterium longum. Deletion of this pneumococcal open reading frame confirmed that this gene encodes an O-glycosidase. Experiments using a model glycoconjugate demonstrated that this O-glycosidase, together with the neuraminidase NanA, is required for S. pneumoniae to cleave sialylated core-1 O-linked glycans. The ability of the O-glycosidase mutant to cleave this glycan structure was restored by both genetic complementation and the addition of O-glycosidase. The mutant showed a reduction in adherence to human airway epithelial cells and a significantly decreased ability to colonize the upper respiratory tract, suggesting that cleavage of core-1 O-linked glycans enhances the ability of S. pneumoniae to colonize the human airway.